Biology Reference
In-Depth Information
concentration at the tip of the microelectrode. During self-referencing, when the
CaSM is positioned near a solid object, released Ca
2
þ
can accumulate between the
CaSM and the object in a short period of time leading to an artificially higher
concentration of Ca
2
þ
in the constrained space. Likewise, uptake of Ca
2
þ
by the
CaSM can lead to a depletion of Ca
2
þ
in the constrained space. These artifacts are
most apparent in solutions of low background [Ca
2
þ
].
Figure 3
shows examples of
both extremes where CaSMs are self-referenced near a 100-
m
m diameter glass
bead. The CaSM is moving in a path so that the plane of its tip is always parallel
to the near surface of the bead to enable the ISM to get closer to the surface. The
ion trapping e
ect is reduced when the path of excursion orients the plane of the tip
of the CaSM perpendicular to the near surface of the solid object, as shown in
Fig. 2
A and B, because the liquid membrane surface cannot get as close to the solid
object. E
V
ux of Ca
2
þ
from the microelectrode tip occurs when constructed with
100 mM CaCl
2
backfilling solution, originally performed by
K ¨htreiber and Ja
Z
e
(1990)
. Accumulation of the released Ca
2
þ
in less than 1 s can be detected when the
bath [Ca
2
þ
]is50
m
M but not when it is 2 mM, giving rise to an artificial e
V
ux of
Ca
2
þ
from the solid glass bead. Reducing the concentration of the primary ion in
the backfilling solution is one method of reducing the ion leak (
Bakker and
Meyerho
Z
, 2000
). However, when used with self-referencing this can lead to an
artifact of the opposite polarity shown in
Fig. 3
. The CaSM constructed with
V
50
100 mM Ca
2+
backfill, 50
M Ca
2+
bath
100 mM Ca
2+
backfill, 2 mM Ca
2+
bath
100 nM Ca
2+
backfill, 50
m
40
M Ca
2+
bath
100 nM Ca
2+
backfill, 2 mM Ca
2+
bath
50
m
30
m
M Ca
2+
backfill, 50
m
M Ca
2+
bath
20
10
0
−
10
−
20
−
30
−
40
0
5
10
15
20
25
30
Distance from bead (
m
m)
Fig. 3
Ca
2
þ
movement across the tip of a CaSM can be detected in low background [Ca
2
þ
] near a
solid object. Electroneutral exchange of Ca
2
þ
out of the tip of a CaSM (filled box) or into the tip of the
CaSM (filled circle) can give rise to accumulation or depletion of the local [Ca
2
þ
] between a solid object
and the tip of the CaSM. In higher bath [Ca
2
þ
] (empty box, empty circle) the accumulation or depletion
is insignificant compared to the background [Ca
2
þ
] and is therefore not detected. In lowered bath [Ca
2
þ
]
careful balancing of the backfilling [Ca
2
þ
] with the bath [Ca
2
þ
] can reduce (filled triangle) if not
eliminate net movement of Ca
2
þ
across the liquid membrane.
